The process of discovering a new therapeutic agents, traditionally involves the following stages: i) identification of a drug target, ii) validation of the target, iii) screening for compounds that affect the activity of the target, iv) testing lead compounds for toxicity, v) testing lead compounds for side effects, and vi) examining the metabolism and stability of lead compounds, in the patient or in an appropriate model system. Once a potential therapeutic target has been identified and validated, the initial stage of drug discovery requires the screening of often hundreds of thousands of compounds to identify those that regulate the target in the appropriate therapeutic manner. This screening process requires the development of assay techniques which can quickly and inexpensively measure the potency of compounds that regulate the target factor of interest. These high-throughput screening assays can take various forms that include either cell-based or biochemical assays that often rely on colorimetric, fluorescence, radiometric or luminescence-based detection in order to measure receptor activation, RNA, protein concentration, enzyme activity or the physical interaction of proteins to form a functional complex. A constant challenge facing the drug discovery field is to increase the speed and efficiency by which potential lead compounds are identified from the tens of thousands of chemical compounds tested in compound library screens, and thereafter optimised into new pharmacological agents. A common problem encountered during lead optimisation is that the drug candidate originally identified by virtue of its ability to modulate the activity of one or a few specific target proteins also often has one or more contra-indications. Detrimental effects can be caused by the lack of specificity of a compound, thus causing the agent to target a broad range of factors and biological processes in addition to the intended target. Other areas of concern include drug toxicity and metabolism, such that compounds that elicit toxic responses can disrupt normal cellular and tissue function and lead to cell death. Certain compounds have also been demonstrated to regulate their own metabolism, thereby stimulating the breakdown of the target agent and excretion from the body leading to decreased drug efficacy.
Current high throughput screening assays generally focus on measuring the effectiveness of compounds in regulating the activity of a single factor or target, and rely on extended processes of secondary screening and follow-up analyses in order to determine other characteristics of compound function, such as specificity and toxicity.
This increases the amount of time and cost required to develop and optimize compounds into drugs with high therapeutic indices (i.e. high efficacy, high specificity, low toxicity). As a result, many compounds, originally selected because of their activity on the target, are eventually discarded because of subsequently discovered side effects, resulting in wasted effort on evaluating drug leads which ultimately prove unsatisfactory.
In the process of drug discovery and lead optimisation, there is a requirement for faster, more effective, less expensive and especially information-rich screening assays that provide simultaneous information on various compound characteristics and their affects on various cellular pathways (i.e. efficacy, specificity, toxicity and drug metabolism).
One approach that has been previously taken, for example, is described in US 2005/0164321 (Promega Corp) which describes a method using enzyme-mediated reactions for multiplex luminogenic and non-luminogenic assays in the same well to detect the amount (e.g., activity) or presence in a sample of one or more moieties, including cofactors for enzymatic reactions such as ATP, proteins (peptides or polypeptides) that bind to and/or alter the conformation of a molecule, e.g., proteins that modify or cleave a peptide or polypeptide substrate, or a molecule which is bound by and/or altered by a protein.
WO 98/58074 (Allelix Biopharma) describes assay methods and compositions useful for screening chemical compounds to identify ligands for receptors including G-protein coupled receptors. The invention employs cells in which a receptor of interest is coupled through a second messenger system to an ion channel that is gated by cyclic nucleotide. Receptor stimulation causes the second messenger system to produce cyclic nucleotide, which results in a measurable ion influx through the channel. The invention also provides a multiplexed system in which mixed cell cultures expressing different receptor types are loaded with different fluorescent reporters of ion influx.
EP1439384 (Cellomics Inc) provides methods and analytical systems for the determination of the distribution, environment, or activity of fluorescently labelled reporter molecules in cells for the purpose of screening large numbers of compounds for those that specifically affect particular biological functions.
Bertelsen, M., (Methods in Enzymology, (2006), 414, 348-363) describes multiplex analysis of inflammatory signalling with intracellular protein translocation using a high-content imaging system.
Howell, B. J. et al. (Methods in Enzymology, (2006), 414, 284-300, 2006) describe the development and implementation of multiplexed cell-based imaging assays for monitoring cell proliferation, cell cycle stage and apoptosis employing fluorescence microscopy.
Nickischer, D. et al. (Methods in Enzymology, (2006), 414, 389-418) describe the development and implementation of three mitogen-activated protein kinase (MAPK) intracellular signalling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-JUN and ERK activation.
Hanson, B. et al. (J. Biomolecular Screening, (2006), 11, 644-651) describe multiplex intracellular assays through the combination of a fluo-4 calcium mobilisation assay and the beta lactamase reporter system, enabling two G-protein coupled receptor assays drug screens with one cell line.
Jonas, J-C. et al. (Diabetes, (1998), 47, 1266-1273) describe the temporal and quantitative relationship between intracellular Ca2+ concentration and extracellular insulin secretion from a cellular perfusate of isolated pancreatic islet cells stimulated with glucose. Cultured islets were loaded with the calcium indicator fura-PE3 in a medium containing glucose and one islet transferred to a perfusion chamber. The [Ca2+] was measured by fluorescence microscopy, while insulin was determined by RIA from fractions collected downstream from the perfusate. This publication therefore describes the relationship between [Ca2+] and insulin secretion from a large, mixed (i.e. heterogeneous) population of cells, and thus there is no specific correlation between cell stimulation and associated analyte production at the single cell level. Furthermore, Jonas et al does not describe multiplexed assays or the measurement of cell-associated molecules/analytes.
Marriott et al. (J. Cellular Physiology, 1998, 177, 2, 232-240) describe induction of interleukin-6 mRNA expression and cellular calcium measurements in murine peritoneal macrophages. Both mRNA and cellular calcium measurements are intracellular events as mRNA is not secreted by the cell.
Veronesi et al. (Neurotoxicology, 2003, 24, 463-473) describe intracellular calcium and extracellular IL-6 measurements from broncheal-tracheal epithelial cells. In this paper, IL-6 is not cell associated and is measured downstream by ELISA. The measurement of intracellular calcium and extracellular IL-6 measurements from human monocytes by downstream radioimmunoassay, which are carried out without a cellular washing step, is described by McMillen et al. (Critical Care Medicine, 1995, 23, (1) 34-40).
Drucker et al. (Blood, 2002, 100 (11) Abstract number 5025) report intracellular calcium and extracellular IL-6 measurements from multiple myeloma cell lines by downstream ELISA without a cellular washing step.
The measurement of intracellular calcium and neuropeptide Y (NPY) immunochemical staining in the same population of cells is disclosed by Kohno et al. (Diabetes, 2003, 52, (4) 948-956). NPY was detected following cellular fixation with 4% paraformaldehyde on non-living cells.
Lundgren et al. (World Journal of Surgery, 1996, 20, (7) 727-735) describe intracellular calcium and extracellular PTH measurements from human parathyroid cells by downstream radioimmunoassay, without use of a cellular washing step.
None of the above methods give temporal and multiplexed, high content information from stimulated live cells where intracellular and cell-associated signalling factors are measured in a cell from a single homogeneous population of cells. The above methods are therefore unable to correlate specific intracellular events and the downstream production of cell-associated analytes. The present invention addresses these limitations as well as providing numerous advantages over known methods.